Neurotransmitters and hormones rely upon G proteins to exert their effects on target cells. In contrast, growth factor tyrosine kinase receptors recruit a cascade of linked signaling proteins including ras-like GTPases to regulate cell growth and differentiation. Recent compelling evidence indicates that additional novel signaling proteins, including certain regulators of G_. protein signaling (RGS proteins) integrate G protein and ras superfamily-directed pathways at many levels. RGS proteins are highly diverse, multifunctional proteins that bind directly to G proteins to regulate their functions. RGSI4 contains domains (RGS, GoLoco and RID domains) that we and others have shown to interact with active Gcd/o-GTP (RGS domain), inactive Gcd/o-GDP (GoLoco domain) and with the ras-like GTPases rapl and rap2 (RID domain). Activation of rapl/2 pathways in host cells stimulates MAPK/Erk kinase signaling and neuronal differentiation. Roles for RGS14 as a dual regulator of Gai and Gao signaling are not known. Furthermore, how RGS14, Gcd/o and rapl/2 interact to regulate one anothers functions, and what cellular mechanisms are involved in regulating these interactions and linked cellular responses is not known. Our preliminary studies indicate that RGS14 is phosphorylated by kinases involved in G protein and rap signaling. Our hypothesis is that RGS14 is an integrator of G protein (Ged/o) and rap-directed signaling pathways, and that stimulus-initiated phosphorylation regulates RGS 14 interactions with G_ and rap, its subeellular localization, and its signaling functions. Using modern molecular, cellular and biochemical approaches, the specific aims will be to: 1. Determine roles for RGS I4 as a bifunctional regulator of receptor and God and G(_o signaling in membranes. 2. Define cellular and biochemical mechanisms that regulate RGS 14 membrane recruitment and attachment, subcellular localization, and a-Ssociati0n with mChoR, Gczi/o, and rapl/2 in host B35 cells. 3. Define cellular mechanisms for regulating RGS14 phosphorylation in cells, and roles for phosphorylation on RGS14 interactions with mChoR/Gi/o and rapt/2 in vitro, and linked signaling pathways in B35 cells. 4. Determine the effects of overexpressing RGSI4 or eliminating native RGS14 in B35 cells on mChoR/Gi/o and rapl/2- directed signaling events and cellular responses. Impact: These studies will identify previously unrecognized cell signaling roles for RGS proteins, and provide important information about novel mechanisms for cross-talk between G protein signaling pathways and rap/MAPkinase signaling pathways. Findings from these studies will clarify our understanding of the complexity of G protein/RGS protein signaling, and identify potential new targets for therapeutic intervention. PERFORMANCESITE(S) (organization,city,state) Department of Pharmacology Emory University School of Medicine Atlanta, GA 30322 KEYPERSONNEL. Seeinstructions. Use continuationpagesas neededto providethe requiredinformationin theformat shownbelow. Startwith PrincipalInvestigator.List allother key personnelin alphabeticalorder,last namefirst. Name Organization Role on Project John R. Hepler, Ph.D. Emory University Principal Investigator Richard Peavy, Ph.D. Emory University Postdoctoral Fellow Susanne Hollinger Emory University Graduate Student Kate Hubbard Emory University Graduate Student Suneela Ramineni Emory University Research Specialist Allan I. Levey, MD, PhD Emory University Collaborator Jan Pohl, Ph.D Emory University Collaborator Peter, Chidiac, Ph.D. Univ. Western Ontario Collaborator Keith Berland, Ph.D. Emory University Collaborator Richard Neubig, Ph.D. Univ. of Michigan, Ann Arbor Collaborator DisclosurePermissionStatement. Aoolicableto SBIR/STTROnly. See instructions. [] Yes [] No _ PHS398 (Rev.05/01) Page_2 Form Page 2 _ [] Principal InvestigatodProgram Director (Last, first, middle): Hepler, John R. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,